Process for the purification of serum albumin

ABSTRACT

Recombinantly produced serum albumin is purified in a series of steps, optionally by incubation with an anion-exchange adsorbent, followed by affinity chromatography employing a hydrophobic solid phase and using a water-soluble lipid anion as desorbens in the aqueous phase. This immobile phase comprises a carrier coupled to a 2-mercapto or 2-hydroxy alkanoic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/286,406, filed Aug. 5,1994, now abandoned, which is a continuation of application Ser. No.08/030,214, filed as PCT/NL92/00125, Jul. 10, 1992, published asWO93/01207, Jan. 21, 1993, now abandoned.

INTRODUCTION TECHNICAL FIELD

The field concerns purification of serum albumins, particularly humanserum albumins.

BACKGROUND AND RELEVANT LITERATURE

Human serum albumin (HSA) is the major protein component of plasma. Theprimary function of albumin in plasma is maintenance of the colloidosmotic pressure within the blood vessel. Furthermore, the protein actsas a carrier of several ligands, for instance bilirubin and fatty acids.(See reviews by F. Rothstein, V. M. Rosenoer and W. L. Hughes in AlbuminStruct. Funct. Uses (1977) 7-25; U. Kragh-Hansen, Pharmacol. Rev. (1981)33:17-53; T. Peters Jr., Adv. Prot. Chem. (1985) 37:161-245).

Purified serum albumin is indicated for the prevention and treatment ofhypovolemic shock, in conditions where there is severe hypoalbuminemia,as an adjunct in haemodialysis and in cardiopulmonary bypass proceduresand in conjunction with exchange transfusion in the treatment ofneonatal hyperbilirubinemia.

Since large amounts of serum albumin are necessary for therapy and thesource of serum albumin (plasma) is limited, other techniques have beensought to produce HSA in large quantities. Successes have been reportedin the production of HSA by fermentation using transformedmicroorganisms or cell lines made by recombinant DNA techniques. See,for example, EP-A-0073646.

However, one of the major problems in the purification of serum albuminproduced by fermentation using transformed cells is the presence ofcontaminating components from the growth medium (fermentation broth) orcell lysate, which have to be removed in order to obtain purified,homogeneous serum albumin.

In EP-A-0361991 the purification of HSA produced with transformed yeast,using techniques known in the art, yields a product of more than 99%purity. For a pharmaceutical preparation a higher purity is desirable.

Recently a process for the purification of serum albumins based on aprocess in three steps was disclosed in EP-A-0319067. This processstarting with an alkaline precipitation step followed by anion-exchangechromatography and finally affinity chromatography yields a product witha good yield and purity. In EP-A-0319067 a BrCN-activated Sepharose 4Bsupport was mentioned which was prepared according to the methoddescribed by Wichman and Andersson (1974). For industrial use theaffinity matrix based on BrCN-activated sepharose has two majordrawbacks.

First, the isourea linkage (M. Wilchek, T. Miron and J. Kohn in: Methodsin Enzymology (1984) 104:3, W. B. Jakoley Ed., Academic Press, London)resulting from the reaction with the primary alkylamine (spacer) has apositive charge under physiological conditions. This charged spacershows anion-exchange like characteristics that might interfere with thebiospecific adsorption. A second disadvantage is the limited stabilityof the isourea linkage under slightly alkaline conditions (C. M. Yangand G. T. Tsao in: Ad. Biochem. Engin. (1982) 25:19, A. Fiechter Ed.,Springer Verlag, Berlin-Heidelberg) which enables the use of this matrixunder sanitizing conditions (0.1-2.0M NaOH) necessary for the productionof pharmaceutical products.

Therefore, there is a great need for a practical process for large-scalepurification of human serum albumin with a high recovery and a very highpurity.

SUMMARY OF THE INVENTION

Human serum albumin, produced by a transformed host, is purified byion-exchange chromatography, followed by affinity chromatographyemploying a lipophilic surface immobile phase comprising a carriercoupled to a 2-mercapto or 2-hydroxy C4-C14 alkanoic acid, or salt orester thereof. The serum albumin degradation products present at the endof the fermentation which strongly resemble the mature, intact albuminare selectively removed by the fatty acid affinity chromatographyapplied. High recovery and extremely high purity are achieved.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: SDS-PAGE under reducing conditions (CBB R-250 stain). Molecularweight markers: lanes 1 and 5 (24 μg). Purified recombinant HSA afterQ-sepharose and affinity chromatography: lane 3 (30 μg). Commerciallyavailable from human blood derived HSA (Sigma human albumin No. A-8763:lot: 109F-9304): lane 4 (30 μg).

FIG. 2: High performance size-exclusion chromatography on BiosilTSK-250® of purified recombinant HSA.

FIG. 3: High performance size-exclusion chromatography on BiosilTSK-250® of commercially available from human blood derived HSA.

FIG. 4: High performance ion-exchange chromatography on Mono Q® ofpurified recombinant HSA after Q-sepharose and affinity chromatography.

FIG. 5: High performance ion-exchange chromatography on Mono Q® ofcommercially available from human blood derived HSA after Q-sepharoseand affinity chromatography.

FIG. 6: Isoelectrofocussing (CBB R-250 stain). Markers (lanes 1 and 4),purified recombinant HSA after Q-sepharose and affinity chromatography(lane 2 (20 μg)) and commercially available from human blood derived HSA(lane 3 (20 μg)).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the subject invention, human serum albumin, preparedby fermentation using transformed hosts, e.g. microorganisms or celllines, made by recombinant DNA techniques, is purified with highrecovery and in high purity for use as a pharmacological product. Thepurification method is preceded by centrifugation and ultrafiltration ofa fermentation broth. The method can be applied to the thus obtainedsupernatant or to the cells after lysis. It generally involves thefollowing steps:

binding of the serum albumin under slightly acidic conditions to ananion-exchange resin; the albumin is desorbed from the resin by loweringthe pH of the eluens;

next, optionally, ultrafiltration of the medium;

finally, the essential chromatographic step consisting of affinitychromatography employing a lipophilic immobile phase composed of ahydrophilic matrix, spacer and fatty acid derivative as ligand and alipophilic anion as desorbens in the eluens.

The serum albumin may then be harvested by desalting and concentration.The serum albumin produced by the subject process is substantiallyhomogeneous and monomeric, viz. more than 99.95% pure.

The method finds use with the production of serum albumin, particularlyhuman serum albumin, prepared by recombinant techniques employingmicroorganisms or cell lines. The microorganisms may be prokaryotic oreukaryotic, particularly eukaryotic, and include bacteria such as E.coli, B. subtilis, B. licheniformis, Streptomyces, Pseudomonas, etc.

Among eukaryotes are yeasts, such as Saccharomyces, Schizosaccharomyces,Kluvveromyces, Candida, etc., filamentous fungi, Neurospora,Aspergillus, etc.

The expression of the serum albumin may result in secretion or retentionof the product in the organism. The broth may be removed batch-wise orcontinuously from the fermenter. In the case of secretion, the cell-freesupernatant is used in the purification process. The cell-freesupernatant is obtained by clarification of the fermentation broth,conveniently by centrifuging and/or filtering the broth, usingultrafiltration for concentration of the protein product. The filterwill generally have a cut-off of 500-100,000 D, more usually at leastabout 10,000 D.

Where the product or part thereof is retained in the cytoplasm of thecell, the cells are harvested. A lysate may be produced in accordancewith any convenient technique, using mechanical or chemical disruptionof the cells to produce the lysate. The cellular debris may be removedby centrifugation.

The pH of the cell-free supernatant or cell lysate is adjusted to about5 to 9, more preferably to about 6 to 6.5. The pH may be modified by anyconvenient means, such as the addition of sodium hydroxide, or otherconvenient base, usually at a normality in the range of about 0.1 toconcentrated.

The next step is anion-exchange chromatography. The cell-freesupernatant with a buffer concentration from about 20 to 200 mM will beapplied to the preconditioned column. The loading of the anion gel willbe from 5 to 50 g of protein per liter gel, more preferably from 15 to25 g of protein per liter gel.

Next the gel can be washed with equilibration buffer. The volume of thewashes is not critical, generally being from 0.5-10 based on the volumeof the gel. The buffer will generally have a conductivity of 0.1-100mS/cm, preferably between about 1 to 10 mS/cm. The albumin is desorbedfrom the gel by eluting with a generally dilute buffer of moderately lowpH. The buffer concentration will be from 10 to 100 mM. Desirably thebuffer will be at a pH in the range of about pH 4.0-6.0, more desirablebetween pH 4.25 and 4.75.

The ion-exchange step removes nucleic acids, endotoxins and the majorpart of the contaminating non-albumin like proteins. Anion-exchangeadsorbents such as QAE or DEAE bound to a commercially available carrierare employed.

The next essential step is affinity chromatography, where a lipophilicimmobile phase is used. The binding of the lipophilic molecules to thegel is performed by using a bifunctional reagent, capable of bothactivating and coupling of the lipophilic molecules. Examples of suchreagent are epoxy compounds, as for instance epihalohydrin, such asepichlorohydrin, or bisoxirans such as an alkanediolether, for instance1,4-butane-dioldiglycidoxyether. The reaction with the gel matrix yieldsderivatives which possess a hydrophilic, reactive epoxide which canreact with nucleophilic ligands such as 2-mercapto or 2-hydroxy C4-C14alkanoic acids. See for more details: Affinity Chromatography: APractical Approach, ed. Dean, P. D. G., Johnson, W. S. and Middle, F.A., IRL Press, Oxford, England, ISBN-0-904147-71-1.

These affinity chromatographic adsorbents are highly stable and do notpossess a charge in the spacer.

After the ion-exchange step the pH of the medium must be increased tophysiologic conditions, normally in the range of about 6.5 to 8.0, moreusually in the range of about 7 to 7.5. The buffer concentration willgenerally be in the range of 50 to 250 mM. After applying the medium tothe column the gel is washed with equilibration buffer. The volume ofthe washes is not critical, generally being from 0.25-5 volume based onthe volume of the gel. The buffer will generally have a conductivity of1.0-20 mS/cm, preferably between about 10 to 15 mS/cm. The loading ofthe affinity gel will be from 5-70 g of protein per liter gel, morepreferably from 15-30 g of protein per liter gel. The albumin is elutedby applying a buffer with a fatty acid with a concentration of 25-250mM, for instance sodium caprylate.

Besides mature human serum albumin, specific degradation products arepresent in the supernatant of the fermentation broth. These degradationproducts have a molecular weight of 40-50 kD and consist of domain I andII of the mature protein that comprises three domains. Domain III absentin said 40-50 kD fragment contains the principle fatty acid binding siteof albumin (T. Peters, supra). Surprisingly, said 40-50 kD fragmentsshow a higher affinity for lipophilic affinity matrix than mature humanserum albumin which enables the separation of the 40-50 kD fragmentsfrom the mature protein.

A wide variety of supports and adsorbents may be used as the solidcarriers or supports. Such solid carriers include inorganic carriers,such as glass and silica gel, organic, synthetic or naturally occurringcarriers, such as, for instance, agarose, cellulose, dextran, polyamide,polyacrylamides, vinyl copolymers of bifunctional acrylates, and varioushydroxylated monomers, and the like. Commercially available carriers aresold under the names of Sephadex®, Sepharose®, Trisacryl®, Ultrogel®,Dynospheres®, Macrosorb®, XAD resins, and others.

The conditions for the various steps will be carried out atnon-denaturing conditions, generally at convenient temperatures in therange of about -10° C. to +30° C., more usually at about ambienttemperatures. The chromatographic steps may be performed batch-wise orcontinuously, as convenient. Any convenient method of separation may beemployed, such as centrifugation, filtration, decanting, or the like. Inthis way a preparation of isolated serum albumin with a surprisinglyhigh purity of more than 99.9% pure, particularly more than 99.95% pure,can be obtained.

All documents cited are incorporated herein by reference.

The following non-limitative examples will further illustrate theinvention.

EXAMPLES Example 1

Synthesis of affinity matrices

E-C10-C10:

E stands for epoxy-activated sepharose 6FF;

first C10 stands for the spacer length of 10 C atoms;

second C10 stands for the number of C atoms in the carboxylic acidligand.

For the synthesis of the affinity matrices well known methods were used(see e.g. Dean et al., supra).

For the preparation of C3 and C10 spacers epichlorohydrin and 1,4butanediolglycidoxyether were used, respectively.

Synthesis of the E-C10-C10 matrix:

1 l Sepharose 6FF was washed thoroughly with distilled water. The excesswater was removed by a sintered glass filter. The gel was suspended in0.6 1 of 1,4 butanediolglycidoxyether and stirred for approximately 30min. Next 0.6 1 of a solution of 0.5N NaOH was added and stirring wascontinued for 24 h. The epoxy-activated gel was thoroughly washed withdistilled water and stored at 0° C. Yield 0.7 kg of wet product (E-C10).

The epoxy-activated sepharose 6FF (0.5 kg of wet product, E-C10) wassuspended in a solution of 8 g 2-mercaptodecanoic acid in a solution of0.5 M sodium carbonate and stirred for 24 h. The gel was filtered on asintered glass filter and washed successively with 0.2M sodium carbonateand water. The wet gel was stored at 0° C. This matrix was calledE-C10-C10.

The number of free carboxylic acids was determined by simple titrationand was found to be 19.3 μequivalents per gram of wet gel matrix.

Example 2

Purification of recombinant HSA from a clarified fermentation broth byQ-sepharose and E-C10-C10-affinity chromatography

Fermentation:

The Kluvveromyces lactis strain CBS 2360 (Centraal Bureau voorSchimmelcultures, Baarn, The Netherlands), transformed with a plasmidcontaining the gene for HSA, was grown for 90 h at 30° C. in a mediumcontaining yeast extract 0.5% (w/v), glucose 1.4% (w/v), caseinhydrolysate 1.0% (w/v), vitamins and mineral salts. During thefermentation glucose was fed.

Filtration and ultrafiltration:

The fermentation broth was centrifuged (5 min at 4000 rpm). Thesupernatant was filtered through an EKS filter and was concentrated 11times by ultrafiltration using a filter with a cut-off of 30,000 D. Theprotein concentration after ultrafiltration was 28 mg/ml, of which 65%is monomeric and mature recombinant HSA. The pH was adjusted to 6.4 byaddition of 0.5M sodium acetate pH 5.5.

Q-sepharose chromatography:

The HSA solution was bound to a Q-sepharose FF column (load: 25 g ofprotein per liter sepharose), which has been equilibrated with 50 mMsodium acetate pH 5.5. After washing the gel with the equilibrationbuffer, the RHA was eluted with 50 mM sodium acetate pH 4.6. The pH ofthe eluate was increased to pH 7.4 by addition of 1M phosphate buffer pH6.6 (ratio eluate:buffer (v/v) =10:1) and 4M NaOH. The proteinconcentration was 10 mg/ml, of which 78% is monomeric and maturerecombinant HSA.

Affinity chromatography:

The HSA containing Q-sepharose eluate was contacted with2-mercaptodecanoic acid coupled to epoxy-activated sepharose 6FF by adiglycidylether (E-C10-C10-sepharose) as described in Example 1. Theaffinity absorbent was equilibrated with 100 mM sodium phosphate bufferpH 7.4. After binding the recombinant HSA to the absorbent (load: 22 gof protein per liter gel) the column was washed with the equilibrationbuffer. The HSA was eluted with 100 mM sodium phosphate pH 7.4containing 75 mM sodium caprylate. The HSA was desalted and concentratedby ultrafiltration using a filter with a cut-off of 30,000 D. Thepurified HSA had a protein concentration of 55 mg/ml, of which more than99.9% was monomeric and mature HSA.

In this example the recovery of HSA was 50%. No contaminants and onlytrace amounts of degradation products could be detected by SDS-PAGE(FIG. 1), HPLC-SEC (FIGS. 2+3), HPLC-IEC (FIGS. 4+5) and IEF (FIG. 6).The purity according to HPLC-SEC was found to be 99.3%. Correction forthe salt related peak at 24.77 gives a purity of>99.91%. In fact, usingimmunoblotting techniques the purity of the purified HSA was estimatedto be at least 99.95%. For this technique polyclonal antibodies wereraised against proteins, which were purified from a blank K. lactisfermentation broth according to the method described in this example.The level of endotoxin was lower than 1 endotoxin unit per ml 5% (w/v)recombinant HSA concentrate. Endotoxin units are related to thereference standard (RSE) delivered by the FDA, the so-called EC-5.Endotoxin was mainly removed during the Q-sepharose column step, seeTable 1.

                  TABLE 1                                                         ______________________________________                                        removal of endotoxin during purification of recombinant                       human serum albumin                                                           Composition       Total endotoxin units                                       ______________________________________                                        K. lactis supernatant                                                                           115,000                                                     effluent Q-sepharose                                                                            113,000                                                     eluate Q-sepharose                                                                              59                                                          effluent affinity-matrix                                                                        41                                                          eluate affinity-matrix                                                                          41                                                          end product (5.5% (w/v) HSA)                                                                    17                                                          ______________________________________                                    

Example 3A

Purification of recombinant HSA from a clarified fermentation broth byQ-sepharose and E-C10-C8-affinity chromatography

Fermentation, filtration and ultrafiltration: performed as described inExample 2.

Q-sepharose chromatography: performed as described in EP-A-0319067.

The collected supernatants were concentrated by ultrafiltration using afilter with a cut-off of 30,000 D. The pH of the retentate was adjustedto pH 7.4. The concentration of monomeric HSA was 14 mg/ml.

Affinity chromatography with E-C10-C8-matrix:

The concentrated Q-sepharose eluate was added to C8 (2-mercaptooctanoicacid) coupled to epoxy-activated sepharose 6FF by a diglycidylether. Theaffinity absorbent was equilibrated and washed after absorption with 100mM sodium phosphate buffer 7.4. The monomeric HSA and degradationproducts could be eluated by 100 mM sodium caprylate. Intact HSA anddegradation products could be separated by using a gradient of 0-100 mMsodium caprylate in 100 mM sodium phosphate buffer pH 7.4. Binding ofHSA and degradation products to the E-C10-C8-matrix was less strong thanbinding to the E-C10-C10-matrix as described in Example 2.

Example 3B

Purification of recombinant HSA from a clarified fermentation broth byQ-sepharose and E-C3-C12-affinity chromatography

Fermentation, filtration, ultrafiltration and Q-sepharosechromatography: performed as described in Example 3A.

Affinity chromatography with E-C3-C12-matrix:

The purification of HSA was performed as described in Example 3A, except2-mercaptododecaoctanoic acid was coupled to epoxy-activated sepharose6FF by C3-ether. Bound monomeric and mature HSA, degradation products ofHSA and other contaminations could not be eluted by 100 mM sodiumcaprylate in 100 mM sodium phosphate buffer pH 7.4. However, elution waspossible with 1% SDS. Separation of intact HSA and other proteins wasnot possible.

We claim:
 1. A method for producing purified recombinant serum albuminsubstantially free of albumin degradation products, said methodcomprising the steps of:(a) incubating at about physiologic pH a productmedium comprising a clarified fermentation broth or clarified celllysate comprising recombinant serum albumin with an anion-exchangeagent; (b) eluting said anion-exchange agent with a buffer solutionhaving an acid pH and collecting first eluate fractions which comprisesaid recombinant serum albumin; (c) contacting at about physiologic pHsaid first eluate fractions with a chromatography system comprising alipophilic immobile phase and an aqueous mobile phase, wherein saidimmobile phase comprises a carrier activated by an epoxide compoundcoupled to a 2-mercapto C4-C14 alkanoic compound or 2-hydroxy C4-C14alkanoic compound selected from the group consisting of an acid, a saltand an ester of either of said compounds; (d) eluting recombinant serumalbumin from said chromatography system by adding as desorbens a watersoluble lipid anion to said aqueous mobile phase and collecting secondeluate fractions comprising recombinant serum albumin; and (e) isolatingfrom said second eluate fractions purified recombinant serum albuminsubstantially free of albumin degradation products.
 2. The methodaccording to claim 1, wherein said epoxide compound is an epoxyether. 3.The method according to claim 2, wherein said epoxyether is adiglycidylether.
 4. A method according to any one of claims 1, 2, or 3,further comprising the step of dialyzing said purified recombinant serumalbumin.
 5. A method according to claim 4, wherein said purifiedrecombinantly produced serum albumin is purified recombinant human serumalbumin.
 6. A method according to any one of claims 1, 2, or 3, whereinsaid purified recombinant serum albumin is purified recombinant humanserum albumin.
 7. A method for producing purified recombinant serumalbumin, said method comprising the steps:(a) incubating at aboutphysiologic pH a product medium comprising a clarified fermentationbroth or clarified cell lysate comprising recombinant serum albumin withan anion-exchange agent; (b) eluting said anion-exchange agent with abuffer solution having an acid pH and collecting first eluate fractionswhich comprise said recombinant serum albumin; (c) contacting at aboutphysiologic pH said first eluate fractions with a chromatography systemcomprising a lipophilic immobile phase and an aqueous mobile phase,wherein said immobile phase comprises a carrier activated by an epoxidecompound coupled to a 2-mercapto C4-C14 alkanoic compound or 2-hydroxyC4-C14 alkanoic compound selected from the group consisting of an acid,a salt and an ester of either of said compounds; (d) eluting recombinantserum albumin from said chromatography system by adding as desorbens awater soluble lipid anion to said aqueous mobile phase and collectingsecond eluate fractions comprising recombinant serum albumin; and (e)isolating from said second eluate fractions purified recombinant serumalbumin substantially free of cellular components of microorganisms orcell lines.
 8. The method according to claim 7, wherein said epoxidecompound is an epoxyether.
 9. The method according to claim 7, whereinsaid epoxyether is a diglycidylether.
 10. A method according to any oneof claims 7, 8 or 9, further comprising the step of dialyzing saidpurified recombinant serum albumin.
 11. A method according to claim 10,wherein said purified recombinant serum albumin is purified recombinanthuman serum albumin.
 12. A method according to any one of claims 7, 8,9, wherein said purified recombinant serum albumin is purifiedrecombinant human serum albumin.
 13. A method for purifying recombinantserum albumin substantially free of albumin degradation products, saidmethod comprising:isolating purified recombinant serum albumin from anaffinity chromatography system, wherein said affinity chromatographysystem comprises a lipophilic immobile phase and an aqueous mobilephase, wherein said lipophilic immobile phase comprises a carrieractivated by an epoxide compound coupled to a 2-mercapto C4-C14 alkanoiccompound or 2-hydroxy C4-C14 alkanoic compound selected from the groupconsisting of an acid, a salt and an ester of either of said compounds.14. A method according to claim 13, wherein said isolating is from aneluate obtained by adding as desorbens a water soluble lipid anion tosaid aqueous mobile phase.
 15. A method according to claim 13, whereinsaid recombinant serum albumin is recombinant human serum albumin.